Inhibition of mutant BRAF splice variant signaling by next‐generation,selective RAF inhibitors

Vemurafenib and dabrafenib block MEK‐ERK1/2 signaling and cause tumor regression in the majority of advanced‐stage BRAF^V600E melanoma patients; however, acquired resistance and paradoxical signaling have driven efforts for more potent and selective RAF inhibitors. Next‐generation RAF inhibitors, such as PLX7904 (PB04), effectively inhibit RAF signaling in BRAF^V600E melanoma cells without paradoxical effects in wild‐type cells. Furthermore, PLX7904 blocks the growth of vemurafenib‐resistant BRAF^V600E cells that express mutant NRAS. Acquired resistance to vemurafenib and dabrafenib is also frequently driven by expression of mutation BRAF splice variants; thus, we tested the effects of PLX7904 and its clinical analog, PLX8394 (PB03), in BRAF^V600E splice variant‐mediated vemurafenib‐resistant cells. We show that paradox‐breaker RAF inhibitors potently block MEK‐ERK1/2 signaling, G1/S cell cycle events, survival and growth of vemurafenib/PLX4720‐resistant cells harboring distinct BRAF^V600E splice variants. These data support the further investigation of paradox‐breaker RAF inhibitors as a second‐line treatment option for patients failing on vemurafenib or dabrafenib.     

Systematic analysis of BRAFV600E melanomas reveals a role for JNK/c-Jun pathway in adaptive resistance to drug-induced apoptosis

Drugs that inhibit RAF/MEK signaling, such as vemurafenib, elicit profound but often temporary anti-tumor responses in patients with BRAF^V600E melanoma. Adaptive responses to RAF/MEK inhibition occur on a timescale of hours to days, involve homeostatic responses that reactivate MAP kinase signaling and compensatory mitogenic pathways, and attenuate the anti-tumor effects of RAF/MEK inhibitors. We profile adaptive responses across a panel of melanoma cell lines using multiplex biochemical measurement, single-cell assays, and statistical modeling and show that adaptation involves at least six signaling cascades that act to reduce drug potency (IC₅₀) and maximal effect (i.e., E_max ≪ 1). Among these cascades, we identify a role for JNK/c-Jun signaling in vemurafenib adaptation and show that RAF and JNK inhibitors synergize in cell killing. This arises because JNK inhibition prevents a subset of cells in a cycling population from becoming quiescent upon vemurafenib treatment, thereby reducing drug E_max. Our findings demonstrate the breadth and diversity of adaptive responses to RAF/MEK inhibition and a means to identify which steps in a signaling cascade are most predictive of phenotypic response.     

RAF protein-serine/threonine kinases: Structure and regulation

A-RAF, B-RAF, and C-RAF are a family of three protein-serine/threonine kinases that participate in the RAS-RAF-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including apoptosis, cell cycle progression, differentiation, proliferation, and transformation to the cancerous state. RAS mutations occur in 15-30% of all human cancers, and B-RAF mutations occur in 30-60% of melanomas, 30-50% of thyroid cancers, and 5-20% of colorectal cancers. Activation of the RAF kinases requires their interaction with RAS-GTP along with dephosphorylation and also phosphorylation by SRC family protein-tyrosine kinases and other protein-serine/threonine kinases. The formation of unique side-to-side RAF dimers is required for full kinase activity. RAF kinase inhibitors are effective in blocking MEK1/2 and ERK1/2 activation in cells containing the oncogenic B-RAF Val600Glu activating mutation. RAF kinase inhibitors lead to the paradoxical increase in RAF kinase activity in cells containing wild-type B-RAF and wild-type or activated mutant RAS. C-RAF plays a key role in this paradoxical increase in downstream MEK-ERK activation.     

The Dual RAF/MEK Inhibitor CH5126766/RO5126766 May Be a Potential Therapy for RAS-Mutated Tumor Cells

Although melanoma is the most aggressive skin cancer, recent advances in BRAF and/or MEK inhibitors against BRAF-mutated melanoma have improved survival rates. Despite these advances, a treatment strategy targeting NRAS-mutated melanoma has not yet been elucidated. We discovered CH5126766/RO5126766 as a potent and selective dual RAF/MEK inhibitor currently under early clinical trials. We examined the activity of CH5126766/RO5126766 in a panel of malignant tumor cell lines including melanoma with a BRAF or NRAS mutation. Eight cell lines including melanoma were assessed for their sensitivity to the BRAF, MEK, or RAF/MEK inhibitor using in vitro growth assays. CH5126766/RO5126766 induced G1 cell cycle arrest in two melanoma cell lines with the BRAF V600E or NRAS mutation. In these cells, the G1 cell cycle arrest was accompanied by up-regulation of the cyclin-dependent kinase inhibitor p27 and down-regulation of cyclinD1. CH5126766/RO5126766 was more effective at reducing colony formation than a MEK inhibitor in NRAS- or KRAS-mutated cells. In the RAS-mutated cells, CH5126766/RO5126766 suppressed the MEK reactivation caused by a MEK inhibitor. In addition, CH5126766/RO5126766 suppressed the tumor growth in SK-MEL-2 xenograft model. The present study indicates that CH5126766/RO5126766 is an attractive RAF/MEK inhibitor in RAS-mutated malignant tumor cells including melanoma.     

Mechanism and inhibition of BRAF kinase

The role of BRAF in tumor initiation has been established, however, the precise mechanism of autoinhibition has only been illustrated recently by several structural studies. These structures uncovered the basis by which the regulatory domains engage in regulating the activity of BRAF kinase domain, which lead to a more complete picture of the regulation cycle of RAF kinases. Small molecule BRAF inhibitors developed specifically to target BRAF^V600E have proven effective at inhibiting the most dominant BRAF mutant in melanomas, but are less potent against other BRAF mutants in RAS-driven diseases due to paradoxical activation of the MAPK pathway. A variety of new generation inhibitors that do not show paradoxical activation have been developed. Alternatively, efforts have begun to develop inhibitors targeting the dimer interface of BRAF. A deeper understanding of BRAF regulation together with more diverse BRAF inhibitors will be beneficial for drug development in RAF or RASdriven cancers.     

Regulation of growth and survival of activated T cells by cell-transducing inhibitors of Ras

We describe the development of cell-penetrating inhibitors of Ras and study their ability to inhibit T cell activation. The inhibitors transduced T cells in a time and concentration-dependent manner and interacted with endogenous Ras. Anti-CD3/CD28-activated cells when treated with the inhibitors, exhibited a notable reduction in cell size, diminished proliferative capacity, and were more prone to apoptosis. Similarly, lymphocytes activated by antigen in vivo, exhibited accelerated apoptosis when treated with the inhibitors ex vivo. Our data reveal a pro-survival role for Ras in activated primary T cells and describe a new methodology for regulating its activity.

Structured summary

MINT-6802882: RAF1 (uniprotkb:P04049) physically interacts (MI:0218) with RAS (uniprotkb:P01112) by anti tag co-immunoprecipitation (MI:0007)     

Synergistic antitumour activity of RAF265 and ZSTK474 on human TT medullary thyroid cancer cells

Medullary thyroid cancer (MTC) is an aggressive malignancy responsible for up to 14% of all thyroid cancer‐related deaths. It is characterized by point mutations in the rearranged during transfection (RET) proto‐oncogene. The activated RET kinase is known to signal via extracellular signal regulated kinase (ERK) and phosphoinositide 3‐kinase (PI3K), leading to enhanced proliferation and resistance to apoptosis. In the present work, we have investigated the effect of two serine/threonine‐protein kinase B‐Raf (BRAF) inhibitors (RAF265 and SB590885), and a PI3K inhibitor (ZSTK474), on RET‐mediated signalling and proliferation in a MTC cell line (TT cells) harbouring the RETC634W activating mutation. The effects of the inhibitors on VEGFR2, PI3K/Akt and mitogen‐activated protein kinases signalling pathways, cell cycle, apoptosis and calcitonin production were also investigated. Only the RAF265+ ZSTK474 combination synergistically reduced the viability of treated cells. We observed a strong decrease in phosphorylated VEGFR2 for RAF265+ ZSTK474 and a signal reduction in activated Akt for ZSTK474. The activated ERK signal also decreased after RAF265 and RAF265+ ZSTK474 treatments. Alone and in combination with ZSTK474, RAF265 induced a sustained increase in necrosis. Only RAF265, alone and combined with ZSTK474, prompted a significant drop in calcitonin production. Combination therapy using RAF265 and ZSTK47 proved effective in MTC, demonstrating a cytotoxic effect. As the two inhibitors have been successfully tested individually in clinical trials on other human cancers, our preclinical data support the feasibility of their combined use in aggressive MTC.     

The PP242 mammalian target of rapamycin (mTOR) inhibitor activates extracellular signal-regulated kinase (ERK) in multiple myeloma cells via a target of rapamycin complex 1 (TORC1)/eukaryotic translation initiation factor 4E (eIF-4E)/RAF pathway and activation is a mechanism of resistance

Activation of PI3-K-AKT and ERK pathways is a complication of mTOR inhibitor therapy. Newer mTOR inhibitors (like pp242) can overcome feedback activation of AKT in multiple myeloma (MM) cells. We, thus, studied if feedback activation of ERK is still a complication of therapy with such drugs in this tumor model. PP242 induced ERK activation in MM cell lines as well as primary cells. Surprisingly, equimolar concentrations of rapamycin were relatively ineffective at ERK activation. Activation was not correlated with P70S6kinase inhibition nor was it prevented by PI3-kinase inhibition. ERK activation was prevented by MEK inhibitors and was associated with concurrent stimulation of RAF kinase activity but not RAS activation. RAF activation correlated with decreased phosphorylation of RAF at Ser-289, Ser-296, and Ser-301 inhibitory residues. Knockdown studies confirmed TORC1 inhibition was the key proximal event that resulted in ERK activation. Furthermore, ectopic expression of eIF-4E blunted pp242-induced ERK phosphorylation. Since pp242 was more potent than rapamycin in causing sequestering of eIF-4E, a TORC1/4E-BP1/eIF-4E-mediated mechanism of ERK activation could explain the greater effectiveness of pp242. Use of MEK inhibitors confirmed ERK activation served as a mechanism of resistance to the lethal effects of pp242. Thus, although active site mTOR inhibitors overcome AKT activation often seen with rapalog therapy, feedback ERK activation is still a problem of resistance, is more severe than that seen with use of first generation rapalogs and is mediated by a TORC1- and eIF-4E-dependent mechanism ultimately signaling to RAF.     

Discovery and initial SAR of pyrimidin-4-yl-1H-imidazole derivatives with antiproliferative activity against melanoma cell lines

The synthesis of a novel series of pyrimidin-4-yl-1H-imidazol-2-yl derivatives 7, 8, 9 and their antiproliferative activities against A375P human melanoma cell line and WM3629 cell line were described. Most compounds showed superior antiproliferative activities compared to Sorafenib, the well-known RAF inhibitor. Among them, 7a exhibited potent activities on both cell lines (IC₅₀ = 0.62 and 4.49 μM, respectively) and turned out to be a selective and potent CRAF inhibitor.     

Design,synthesis and biological evaluation of fused naphthofuro[3,2-c] quinoline-6,7,12-triones and pyrano[3,2-c]quinoline-6,7,8,13-tetraones derivatives as ERK inhibitors with efficacy in BRAF-mutant melanoma

Approximately 60% of human cancers exhibit enhanced activity of ERK1 and ERK2, reflecting their multiple roles in tumor initiation and progression. Acquired drug resistance, especially mechanisms associated with the reactivation of the MAPK (RAF/MEK/ERK) pathway represent a major challenge to current treatments of melanoma and several other cancers. Recently, targeting ERK has evolved as a potentially attractive strategy to overcome this resistance. Herein, we report the design and synthesis of novel series of fused naphthofuro[3,2-c]quinoline-6,7,12-triones 3a-f and pyrano[3,2-c]quinoline-6,7,8,13-tetraones 5a,b and 6, as potential ERK inhibitors. New inhibitors were synthesized and identified by different spectroscopic techniques and X-ray crystallography. They were evaluated for their ability to inhibit ERK1/2 in an in vitro radioactive kinase assay. 3b and 6 inhibited ERK1 with IC50s of 0.5 and 0.19 µM, and inhibited ERK2 with IC50s of 0.6 and 0.16 µM respectively. Kinetic mechanism studies revealed that the inhibitors are ATP-competitive inhibitors where 6 inhibited ERK2 with a K_i of 0.09 µM. Six of the new inhibitors were tested for their in vitro anticancer activity against the NCI-60 panel of tumor cell lines. Compound 3b and 6 were the most potent against most of the human tumor cell lines tested. Moreover, 3b and 6 inhibited the proliferation of the BRAF mutant A375 melanoma cells with IC50s of 3.7 and 0.13 µM, respectively. In addition, they suppressed anchorage-dependent colony formation. Treatment of the A375 cell line with 3b and 6 inhibited the phosphorylation of ERK substrates p-90RSK and ELK-1 and induced apoptosis in a dose dependent manner. Finally, a molecular docking study showed the potential binding mode of 3b and 6 within the ATP catalytic binding site of ERK2.     

Residence time and kinetic efficiency analysis of extracellular signal-regulated kinase 2 inhibitors

The RAS/RAF/MEK/ERK signal transduction cascade plays an important role in the regulation of critical cellular processes such as cell proliferation, migration, and differentiation. The up-regulation of this pathway can negatively affect cell homeostasis and is responsible for the development of various forms of cancer and inflammation processes. Therefore, there is a strong interest in pursuing drug programs targeting some of the enzymes involved in this pathway. In addition to the determination of K_i, K_d, IC₅₀, and/or EC_50, a more thorough kinetic analysis can provide useful information for the selection of the best lead series during the early stage of the drug discovery process. This study describes a medium-throughput fluorescent probe displacement assay for the rapid determination of the k_off constant, residence time, and kinetic efficiency for ERK (extracellular signal-regulated kinase) inhibitors. Using this method, we have identified several inhibitors that we have subjected to further kinetic analysis by comparing k_off constants determined for these time-dependent inhibitors using either the active or inactive form of ERK2.     

Loss of PI(4,5)P2 5-Phosphatase A Contributes to Resistance of Human Melanoma Cells to RAF/MEK Inhibitors

Past studies have shown that the inositol polyphosphate 5-phosphatase, phosphatidylinositol 4,5-bisphosphate 5-phosphatase (PIB5PA), is commonly downregulated or lost in melanomas, which contributes to elevated activation of phosphatidylinositol 3-kinase (PI3K)/Akt in melanoma cells. In this report, we provide evidence that PIB5PA deficiency plays a role in resistance of melanoma cells to RAF/mitogen-activated protein kinase kinase (MEK) inhibitors. Ectopic expression of PIB5PA enhanced apoptosis induced by the RAF inhibitor PLX4720 in BRAF^V600E and by the MEK inhibitor U0126 in both BRAF^V600E and wild-type BRAF melanoma cells. This was due to inhibition of PI3K/Akt, as co-introduction of an active form of Akt (myr-Akt) abolished the effect of overexpression of PIB5PA on apoptosis induced by PLX4720 or U0126. While overexpression of PIB5PA triggered activation of Bad and down-regulation of Mcl-1, knockdown of Bad or overexpression of Mcl-1 recapitulated, at least in part, the effect of myr-Akt, suggesting that regulation of Bad and Mcl-1 is involved in PIB5PA-mediated sensitization of melanoma cells to the inhibitors. The role of PIB5PA deficiency in BRAF inhibitor resistance was confirmed by knockdown of PIB5PA, which led to increased growth of BRAF^V600E melanoma cells selected for resistance to PLX4720. Consistent with its role in vitro, overexpression of PIB5PA and the MEK inhibitor selumetinib cooperatively inhibited melanoma tumor growth in a xenograft model. Taken together, these results identify loss of PIB5PA as a novel resistance mechanism of melanoma to RAF/MEK inhibitors and suggest that restoration of PIB5PA may be a useful strategy to improve the therapeutic efficacy of the inhibitors in the treatment of melanoma.     

Proteinase inhibitors from Erythrina corallodendron and Erythrina cristagalli seeds

Eight and five proteinase inhibitors were purified from Erythrina corallodendron and E. cristagalli seeds, respectively, by gel filtration followed by ion exchange chromatography on DEAE-cellulose and DEAE-sepharose. Each inhibitor consists of 161–163 amino acids (M_r 18 000) including four half-cystine residues and resembles the Kunitz-type proteinase inhibitors. The N-terminal amino acid sequence of trypsin inhibitor DE-7 from E. corallodendron seed resembles those of other Erythrina species. For the other inhibitors no free N-terminal amino acid was found. DE-1,-2,-3,-4 and -5 from the seed of E. corallodendron contain potent inhibitors for α-chymotrypsin and they have practically no action on trypsin. From the same seed, inhibitors DE-6, -7 and -8 strongly inhibit trypsin and also inhibit α-chymotrypsin to varying degrees. From the seeds of E. cristagalli, inhibitors DE-1 and -8 inhibit trypsin strongly and DE-2, -3 and -4 are strongly inhibitory for α-chymotrypsin. On summarizing the inhibitor characteristics of the Kunitz-type proteinase inhibitors from the seeds of eight different species of Erythrina, it was obvious that there is a relationship between the alanine content of the inhibitors and their activities. A high alanine content is associated with potent α-chymotrypsin activities and low alanine content with strong trypsin activities.     

Effects of Epichloë gansuensis on root-associated fungal communities of Achnatherum inebrians under different growth conditions

This study was conducted to investigate how Epichloë gansuensis endophyte and soil disturbance affect root-associated fungi (RAF) of Achnatherum inebrians (drunken horse grass, DHG), using Illumina sequencing techniques. The rhizosphere soil of wild endophyte-infected (W-EI) DHG had significantly (P < 0.05) higher available phosphorous and potassium, total organic matter, ammonium and nitrate nitrogen than cultivated soil. In addition, the rhizosphere soil of endophyte-infected DHG had significantly (P < 0.05) lower pH and nitrate nitrogen, and higher available phosphorous, than endophyte-free DHG under cultivated conditions. The sequencing provided a total of 54,413 sequences and these were assigned into 190 operational taxonomic units (OTUs) with 97% similarity. Ascomycota was the most dominant phylum in roots of three DHG populations. W-EI DHG had significantly (P < 0.05) higher RAF diversity than cultivated endophyte-infected (C-EI) DHG. The presence of endophyte significantly (P < 0.05) decreased RAF diversity under cultivation. The principal component analysis (PCA) and sample similarity analysis results indicated that both endophyte and soil disturbance could bring changes to RAF community composition. The RDA results demonstrated the RAF of W-EI DHG were positively correlated with soil properties, and the RAF of cultivated DHG roots were negatively correlated with soil properties. This study demonstrated that both endophyte and soil disturbance resulted in changes to the RAF communities.     

Modulation of Rat Cecal Microbiota by Administration of Raffinose and Encapsulated Bifidobacterium breve

To investigate the effects of administration of raffinose and encapsulated Bifidobacterium breve JCM 1192^T cells on the rat cecal microbiota, in a preclinical synbiotic study groups of male WKAH/Hkm Slc rats were fed for 3 weeks with four different test diets: basal diet (group BD), basal diet supplemented with raffinose (group RAF), basal diet supplemented with encapsulated B. breve (group CB), and basal diet supplemented with both raffinose and encapsulated B. breve (group RCB). The bacterial populations in cecal samples were determined by fluorescence in situ hybridization (FISH) and terminal restriction fragment length polymorphism (T-RFLP). B. breve cells were detected only in the RCB group and accounted for about 6.3% of the total cells as determined by FISH analysis. B. breve was also detected only in the RCB group by T-RFLP analysis. This was in contrast to the CB group, in which no B. breve signals were detected by either FISH or T-RFLP. Increases in the sizes of the populations of Bifidobacterium animalis, a Bifidobacterium indigenous to the rat, were observed in the RAF and RCB groups. Principal-component analysis of T-RFLP results revealed significant alterations in the bacterial populations of rats in the RAF and RCB groups; the population in the CB group was similar to that in the control group (group BD). To the best of our knowledge, these results provide the first clear picture of the changes in the rat cecal microbiota in response to synbiotic administration.     

Molecular Design,Synthesis and Trypanocidal Activity of Dipeptidyl Nitriles as Cruzain Inhibitors

A series of compounds based on the dipeptidyl nitrile scaffold were synthesized and assayed for their inhibitory activity against the T. cruzi cysteine protease cruzain. Structure activity relationships (SARs) were established using three, eleven and twelve variations respectively at the P1, P2 and P3 positions. A K _i value of 16 nM was observed for the most potent of these inhibitors which reflects a degree of non-additivity in the SAR. An X-ray crystal structure was determined for the ligand-protein complex for the structural prototype for the series. Twenty three inhibitors were also evaluated for their anti-trypanosomal effects and an EC₅₀ value of 28 μM was observed for the most potent of these. Although there remains scope for further optimization, the knowledge gained from this study is also transferable to the design of cruzain inhibitors based on warheads other than nitrile as well as alternative scaffolds.     

Alkynamide phthalazinones as a new class of TbrPDEB1 inhibitors

Several 3′,5′-cyclic nucleotide phosphodiesterases (PDEs) have been validated as good drug targets for a large variety of diseases. Trypanosoma brucei PDEB1 (TbrPDEB1) has been designated as a promising drug target for the treatment of human African trypanosomiasis. Recently, the first class of selective nanomolar TbrPDEB1 inhibitors was obtained by targeting the parasite specific P-pocket. However, these biphenyl-substituted tetrahydrophthalazinone-based inhibitors did not show potent cellular activity against Trypanosoma brucei (T. brucei) parasites, leaving room for further optimization. Herein, we report the discovery of a new class of potent TbrPDEB1 inhibitors that display improved activities against T. brucei parasites. Exploring different linkers between the reported tetrahydrophthalazinone core scaffold and the amide tail group resulted in the discovery of alkynamide phthalazinones as new TbrPDEB1 inhibitors, which exhibit submicromolar activities versus T. brucei parasites and no cytotoxicity to human MRC-5 cells. Elucidation of the crystal structure of alkynamide 8b (NPD-048) bound to the catalytic domain of TbrPDEB1 shows a bidentate interaction with the key-residue Gln874 and good directionality towards the P-pocket. Incubation of trypanosomes with alkynamide 8b results in an increase of intracellular cAMP, validating a PDE-mediated effect in vitro and providing a new interesting compound series for further studies towards selective TbrPDEB1 inhibitors with potent phenotypic activity.     

Phosphinyliminodithiolane insecticides: Oxidative bioactivation of phosfolan and mephosfolan

2-(Diethoxyphosphinylimino)-1,3-dithiolane (phosfolan) and its 4-methyl analog (mephosfolan) are proinsecticides as determined by microsomal mixed-function oxidase (MFO) activation to potent acetylcholinesterase (AChE) inhibitors. They are similarly activated by peracid oxidation which yields the sulfoxide and sulfone derivatives. The hydrolytically unstable S-oxides are irreversible AChE inhibitors that are 160- to 47,000-fold more potent than phosfolan and mephosfolan. MFO S-oxidation is indicated for both proinsecticides by (a) NADPH-dependent increases in potency as AChE inhibitors to an extent expected of sulfoxides, and (b) formation of the S-oxide hydrolysis product diethyl phosphoramidate.     

A diverse fungal community associated with Pseudorchis albida (Orchidaceae) roots

In addition to orchid mycorrhizal fungi (OrMF), the roots of orchids harbour plant fungal endophytes termed root-associated fungi (RAF). In the present study, the endangered photosynthetic orchid Pseudorchis albida was screened for OrMF and RAF using culture-dependent (isolations from root sections and pelotons) and culture-independent (cloning from root sections) techniques. The efficiency of the different approaches for detecting the fungi and the effect of the sampling season (summer or autumn) were evaluated. In total, 66 distinct OTUs of mycorrhizal and non-mycorrhizal fungi were found, which, to our knowledge, is the highest diversity of RAF that has yet been detected in a single orchid species. The OrMF community was dominated by Tulasnella species, which were mainly detected by isolation from pelotons or cloning from root sections. The roots and tubers showed higher mycorrhizal colonization in summer, corroborating the frequent reports of Tulasnella from pelotons in this season. In contrast, two helotialean fungi, Varicosporium elodeae and Leohumicola sp., the latter of which was repeatedly isolated from pelotons, were significantly more abundant in the autumn.     

Influence of flavin addition and removal on the formation of superoxide by NADPH-Cytochrome P-450 reductase: a spin-trap study

Several structural derivatives of the dipeptide, l (and d)-cysteinyl-l-proline were synthesized and shown to be very potent competitive and noncompetitive inhibitors of human serum angiotensin I-converting enzyme. Only if the sulfhydryl group of the cysteine was blocked with benzyl, trityl, or benzyloxycarbonyl protecting groups, was the dipeptide a noncompetitive inhibitor. Compounds with free sulfhydryl groups were competitive inhibitors with K_i values in the 10^?8m range. d-Cys-l-Pro, our most potent inhibitor (k₁ = 0.0055 μM), was an order of magnitude more potent than l-Cys-l-Pro consistent with findings of Cushman et al. (1977, Biochemistry16, 5484) that -CH₃ group substitution improves binding if the configuration is d but diminishes binding if the configuration is l. Zinc and calcium ions released inhibition by some of the noncompetitive, but only one, of the competitive inhibitors. The noncompetitive inhibitor, l-cysteinyl(benzyl)-l-proline, and the competitive inhibitor, l-cysteinyl-l-proline, were used as affinity ligands to obtain near homogenous (25 units/mg) enzyme from human plasma. The observation that compounds with a free sulfhydryl group are competitive inhibitors and those in which the sulfhydryl groups are blocked are noncompetitive inhibitors can be rationalized if the active site of the converting enzyme is an extended linear trench.